Paclitaxel, formerly called "Taxol" is an exceptionally promising anticancer agent. It was isolated from the bark of Taxus brevifolia by Wani et al. in 1971 (J. Am. Chem. Soc. 93, 2325, 1971) and its structure was defined using chemical methods and X-ray crystallographic analysis. ##STR1##
Paclitaxel has been approved by the Food and Drug Adminstration for the treatment of breast and ovarian cancer and is currently in clinical trials for the treatment of lung and colon cancers (for example, see W. P. McGuire and E. K. Rowinsky, Paclitaxel in Cancer Treatment, M. Dekker, New York 1995, pages 1 to 337).
A primary natural source for paclitaxel is the bark of the Pacific Yew tree, Taxus brevifolia. It has also been found that paclitaxel is present in the epigeal parts and the roots of other yew species, including the European yew (Taxus baccata), Asian yews (Taxus wallichiana and Taxus chinensis), and yew trees cultivated for ornamental purposes (for example, Taxus media).
The method of isolation of paclitaxel from any natural resource is complex and expensive, partly because of the relatively low concentrations in vegetable materials but also because of the presence of one of its congeners, cephalomannin. The contents of and ratios between paclitaxel and cephalomannin vary in vegetable materials depending on the species and the part of the plant in question. In general, it has been found that the content of paclitaxel and cephalomannin ranges from 0.001% to 0.08% and 0.001% to 0.22% respectively (K. M. Witherup et al., J. Nat. Prod., 53,1249, 1990; R. G. Kelsey et., J. Nat. Prod., 55, 912, 1992; N. C. Wheeler et al., J. Nat. Prod., 55,432, 1992). In particular, the Taxus media species which, being renewable vegetable material, is the most commonly used raw material for the preparation of paclitaxel, contains on the average the highest concentration of cephalomannin in comparison with the other species.
Even the paclitaxel production techniques based on yew cell cultures, which have recently been given a substantial boost to obviate the conventional extraction of expensive vegetable material, yield a relevant quantity of cephalomannin in addition to paclitaxel.
The only structural difference between paclitaxel and cephalomannin involves the side chain portion of the compound, thus giving rise to similar chemical properties. The two compounds, therefore, possess very similar chromatographic properties and clean separation of these related compounds is difficult. A number of chromatographic methods, mainly based on the use of inverted-phase chromatography or expensive bonded-phase columns, have been proposed (J. H. Cardellina, J. Liq. Chromatogr., 12, 2117, 1989), but these cannot be easily adapted to a large commercial scale operation. For this reason, the availability of methods allowing the separation of paclitaxel and cephalomannin remains a topic of great practical importance.
In the past, paclitaxel and cephalomannin separation methods, based on the different reactivity of the two compounds to oxidants, were proposed. It was found that the double olefin bond existing in the tiglic residue of cephalomannin could be oxidized by reaction with osmium tetroxide (D. G. I. Kingston et al. , J. Nat. Prod., 55, 259, 1992) or ozone (J. T. Beckvermit et al., J. Org. Chem., 61, 9038, 1996), while paclitaxel did not undergo any chemical transformation during oxidation reactions. Another approach considered the treatment of mixtures of paclitaxel and cephalomannin with bromine (J. M. Rimoldi et al., J. Nat. Prod., 59, 167, 1996). Treatment with bromine, performed under controlled temperature and time reaction conditions, causes the formation of dibromocephalomannin, while paclitaxel is not affected by this chemical reagent. These methods, however, have a drawback in their use of such toxic reagents as osmium tetroxide and, in any case, result in the destruction or transformation of cephalomannin into its derivatives, from which cephalomannin can be regenerated only through difficult synthetic processes. There is still a need, therefore, for in inexpensive, simple, safe and effective separation of cephalomannin from paclitaxel. Accordingly, the primary objective of this invention is to provide a simple method to separate paclitaxel and cephalomannin from their mixtures or yew extracts.